Xiaojianzhong decoction prevents gastric precancerous lesions in rats by inhibiting autophagy and glycolysis in gastric mucosal cells

BACKGROUND Gastric precancerous lesions(GPL)precede the development of gastric cancer(GC).They are characterized by gastric mucosal intestinal metaplasia and dysplasia caused by various factors such as inflammation,bacterial infection,and injury.Abnormalities in autophagy and glycolysis affect GPL progression,and their effective regulation can aid in GPL treatment and GC prevention.Xiaojianzhong decoction(XJZ)is a classic compound for the treatment of digestive system diseases in ancient China which can inhibit the progression of GPL.However,its specific mechanism of action is still unclear.AIM To investigate the therapeutic effects of XJZ decoction on a rat GPL model and the mechanisms underlying its effects on autophagy and glycolysis regulation in GPLs.METHODS Wistar rats were randomly divided into six groups of five rats each and all groups except the control group were subjected to GPL model construction for 18 wk.The rats’body weight was monitored every 2 wk starting from the beginning of modeling.Gastric histopathology was examined using hematoxylin-eosin staining and Alcian blue-periodic acid-Schiff staining.Autophagy was observed using transmission electron microscopy.The expressions of autophagy,hypoxia,and glycolysis related proteins in gastric mucosa were detected using immunohistochemistry and immunofluorescence.The expressions of the following proteins in gastric tissues:B cell lymphoma/Leukemia-2 and adenovirus E1B19000 interacting protein 3(Bnip-3),microtubule associated protein 1 light chain 3(LC-3),moesin-like BCL2-interacting protein 1(Beclin-1),phosphatidylinositol 3-kimase(PI3K),protein kinase B(AKT),mammalian target of rapamycin(mTOR),p53,AMP-activated protein kinase(AMPK),and Unc-51 like kinase 1(ULK1)were detected using western blot.The relative expressions of autophagy,hypoxia,and glycolysis related mRNA in gastric tissues was detected using reverse transcription-polymerase chain reaction.RESULTS Treatment with XJZ increased the rats’body weight and improved GPL-related histopathological manifestations.It also decreased autophagosome and autolysosome formation in gastric tissues and reduced Bnip-3,Beclin-1,and LC-3II expressions,resulting in inhibition of autophagy.Moreover,XJZ down-regulated glycolysis-related monocarboxylate transporter(MCT1),MCT4,and CD147 expressions.XJZ prevented the increase of autophagy level by decreasing gastric mucosal hypoxia,activating the PI3K/AKT/mTOR pathway,inhibiting the p53/AMPK pathway activation and ULK1 Ser-317 and Ser-555 phosphorylation.In addition,XJZ improved abnormal gastric mucosal glucose metabolism by ameliorating gastric mucosal hypoxia and inhibiting ULK1 expression.CONCLUSION This study demonstrates that XJZ may inhibit autophagy and glycolysis in GPL gastric mucosal cells by improving gastric mucosal hypoxia and regulating PI3K/AKT/mTOR and p53/AMPK/ULK1 signaling pathways,providing a feasible strategy for the GPL treatment.

Enhanced expression of epidermal growth factor receptor gene in gastric mucosal cells by the serum derived from rats treated with electroacupuncture at stomach meridian acupoints

AIM: To investigate the effect of serum derived from rats treated with electroacupuncture at stomach meridian acupoints on the expression of epidermal growth factor receptor (EGFR) gene in gastric mucosal cells. METHODS: The stress-induced gastric mucosal injury in rat model was established by water-immersion and restrained stress methods. 52 rats were randomly divided into: normal group (n = 8), model group (n = 8), model serum group (n = 12), stomach serum group (n = 12), and gallbladder serum group (n = 12). The gastric mucosal cells were separated by pronase-EDTA digestion method and incubated with serum. The EGFR gene expression in gastric mucosal cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: Compared with normal group (0.6860 ± 0.0594), the serum derived from rats of the stomach group (1.2272 ± 0.0813, P = 0.00 < 0.01) and gallbladder group (0.9640 ± 0.0387, P = 0.00 < 0.01) had a tendency to enhance the EGFR gene expression in gastric mucosal cells. Such tendency existed in the model group (0.7104 ± 0.0457) but with no signifi cant difference (P = 0.495 > 0.05) and in model serum group (0.8516 ± 0.0409) with an extremely obvious difference (P = 0.001 < 0.01). Furthermore, the EGFR gene expression in stomach serum group was significantly higher than that in gallbladder serum group (P = 0.00 < 0.01). CONCLUSION: The present study shows that serumderived from rats treated with electroacupuncture at stomach meridian acupoints can distinctly increase the EGFR gene expression of gastric mucosal cells. Therefore, there is certain meridian specificity in the serum, which could provide a proof for the TCM theory “particular relation between meridian and internal organ”.

Cysteamine increases expression and activity of H^1-K^+-ATPase of gastric mucosal cells in weaning piglets

AIM: To determine the in vivo andin vivo effects of cysteamine (CS) on expression and activity of H+-K+-ATPase of gastric mucosal cells in weaning piglets.METHODS: Eighteen litters of newborn Xinhuai piglets were employed in the in vivo experiment and allocated to control and treatment groups. From 12 d of age (D12), piglets in control group were fed basal diet, while the treatment group received basal diet supplemented with 120 mg/kg CS. Piglets were weaned on D35 in both groups. Six piglets from each group (n = 6) were slaughtered on D28 (one week before weaning), D35(weaning), D36.5, D38, D42, and D45 (36 h, 72 h,one week and 10 d after weaning), respectively. Semiquantitative RT-PCR was performed todetermine the levels of H+-K+-ATPase mRNA in gastric mucosa. H+-K+-ATPase activity in gastric mucosa homogenate was also determined. Gastric mucosal epithelial cells from piglets through primary cultures were used to further elucidate the effect of CS on expression and activity of H+-K+-ATPase in vivo. Cells were treated for 20 h with 0.001,0.01, and 0.1 mg/mL of CS (n = 4), respectively. The mRNA expression of H+-K+-ATPase and somatostatin (SS)as well as the H+-K+-ATPase activity were determined.RESULTS: in vivo, both mRNA expression and activity of H+-K+-ATPase in gastric mucosa of control group exhibited a trend to increase from D28 to D45, reaching a peak on D45, but did not show significant age differences. Furthermore, neither the mRNA expression nor the activity of H+-K+-ATPase was affected significantly by weaning. CS increased the mRNA expression of H+-K+-ATPase by 73%, 53%, 30% and 39% on D28(P = 0.014), D35 (P = 0.017), D42 (P = 0.013) and D45(P = 0.046), respectively. In accordance with the mRNA expression, H+-K+-ATPase activities were significantly higher in treatment group than in control group on D35(P = 0.043) and D45 (P = 0.040). In vivo, CS exhibited a dose-dependent effect on mRNA expression and activity of H+-K+-ATPase. Both H+-K+-ATPase mRNA expression and activity in gastric mucosal epithelial cells were significantly elevated after 20 h of exposure to the moderate (H+-K+-ATPase expression: P=0.03; H+-K+-ATPase activity: P = 0.014) and high concentrations (H+-K+-ATPase expression: P=0.017; H+-K+-ATPase activity:P = 0.022) of CS. Significant increases in SS mRNA expression were observed to accompany the elevation of H+-K+-ATPase expression and activity induced by the moderate (P = 0.024) and high concentrations (P = 0.022) of CS. Low concentration of CS exerted no effects either on expression and activity of H+-K+-ATPase or on SS mRNA expression in cultured gastric mucosal epithelial cells.CONCLUSION: No significant changes are observed in mRNA expression and activity of H+-K+-ATPase in gastric mucosa of piglets around weaning from D28 to D45. CS increases expression and activity of gastric H+-K+-ATPase in vivo and in vivo. SS is involved in mediating the effect of CS on gastric H+-K+-ATPase expression and activity in weaning piglets.

Erythropoietin -induced proliferation of gastric mucosal cells

AIM： To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell culture model. METHODS： Erythropoietin receptor was detected by RT-PCR, Western blotting and immunohistochermistry. Growth stimulation effects of erythropoietin on cultured gastric mucosal cells were determined by ELISA using bromodeoxyuridine （BrdU）. RESULTS： Erythropoietin receptor was detected on cultured porcine gastric mucosal epithelial cells. Erythropoietin receptor was also detected histochemically at the base of gastric mucosal epithelium. BrdU assay demonstrated a dose-dependent increase in growth potential of cultured porcine gastric mucosal epithelial cells by administration of erythropoietin, as well as these effects were inhibited by administration of antierythropoietin antibody （P〈 0.01）. CONCLUSION： These findings indicate that erythropoietin has a potential to proliferate gastric mucosal epithelium via erythropoietin receptor.

Effect of bile salts and bile acids on human gastric mucosal epithelial cells

Objective：To explore the effect of bile salt and bile acid on cultured eternalized human gastric mucosa epithelium GES-1 cells. Methods：Cultured eternalized human gastric mucosa epithelium GES-1 cells were treated with media containing 6 different kinds of bile salts and 3 different kinds of bile acids and their mixture with different concentrations： GCDC（glycochenodeoxychoμte）, GDC （glycodeoxychoμte）, GC（glycochoμte）, TCDC（taurochenodeoxychoμte）, TDC（taurodeoxychoμte）, TC （taurochoμte）, LCA （lithocholicacid）, CA（cholic acid）, DCA（deoxycholic acid）（50 μ mol/L,250 μ mol/L,500 μ mol/L,1000 μ mol/L）, DY（mixture of bile salts） and DS（mixture of bile acids）（250 μ mol/L,500 μ mol/L,1000 μ mol/L,1500 μ mol/L, 2000 μ mol/L）, in comparison with the control group（in normal media without bile salts and bile acids）. Cell proliferation was assessed by MTT（3-[4,5-Dimethylthiaolyl]-2,5- diphenyl-tetrazolium bromide） assay for 72 hours with different concentrations and the apoptotic cells were assayed by flow cytometry （FCM） with Annex V-FITC conjugated with propidium iodide（PI） staining for 24 hours with different concentrations（1500,2000 μt mol/L）. Results：There was no significant difference in morphology and cell proliferation in GC group after 24-72 h. Low concentration（50 μ mol/L） of GCDC, GDC, TCDC, TDC and TC accelerated gastric epithelial cell growth in a dosage-time dependent manner. At middle concentration （250-500 μ mol/L）, it showed positive effect after 24-48 h, while negative effect after 72 h. At high concentration（1000 μ mol/L）, it accelerated gastric epithelial cell growth after 24h and show consistent inhibition even leading to necrosis after 48-72 h. LCA and CA showed a positive effect on the concentration of 50 μ mol/L after 24-72 h, while 250-1000 μ mol/L showed a trend towards apoptosis after 24-72 h. At 50-500 μmol/L, DCA showed proliferation after 24 h and apoptosis after 48-72 h, but showed necrosis after 24-72 h at 1000 μmol/L. DY and DS could facilitate normal gastric mucosa epithelial cell growth at low concentration （250-500 μ mol/L）, however at 1000-2000 μ mol/L the trend shifted from apoptosis to necrosis. FCM with Annexin-V conjugated with PI staining revealed that GCDC, GDC, GC, TCDC, TDC, TC, LCA, CA, DCA, DY and DS induced apoptosis of human gastric mucosal epithelial cells. They were all significantly higher than that of the control（P 〈 0.05）, but there was no significant difference in GC group （P 〉 0.05）. The bile salts induced apoptosis in a time-dose-dependent manner. Conclusion：Our results suggested that bile acid and bile salt is the trigger of injury in human gastric mucosal epithelial cells.

Telomerase and hTERT: Can they serve as markers for gastric cancer diagnosis?

AIM: To investigate telomerase activity and human telomerase reverse transcriptase (hTERT) expression in normal human gastric mucosal epithelial cells (nhGMECs) and fibroblasts (nhGMFs).